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Previous studies on the intracellular force balance that forms the adherent cell structure have paid much attention to the mechanical behavior of cells seen in the horizontal project plane. By contrast, there are only few quantitative considerations on that in the vertical plane. Particularly, the contribution of the nucleus to the bearing of the vertical cell structure remains unclear. Here, we investigated the determinant of the vertical cell morphology from experimental and numerical approaches. The effect of cytoskeleton-affecting agents on the vascular endothelial cell height, as a measure of the vertical force balance, was examined by atomic force microscope indentation, demonstrating that actin depolymerization caused an increase in cell height. In contrast, disruption of microtubules lowered the cell height, whereas their stabilization elevated the cell plasma membrane. Time-lapse microscopy showed that intracellular vesicles moved radially outward after the microtubule disruption, together with an enlargement of the nuclear area in the project plane, that is probably associated with the decrease in cell height. Finite element analyses employing a 3D model were carried out to interpret the experimental results and examine potent parameters (such as prestress, elastic modulus, and Poisson's ratio) that affect vertical cell morphology. How the prestress in subcellular components influences cells subjected to extracellular tensile forces was also examined. These results indicate that the nuclear/cytoplasmic mechanical properties and degrees of prestress determine the vertical section structure of adhering cells.

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.

Window setting is a very important technique in CT examinations. However, most beginner technologists have difficulty in setting the optimal window. Now, thanks to technical progress, it is easy to obtain a great many CT images. On the other hand, it is impossible to provide the optimal window setting for all images. Therefore, our purpose is to offer optimal CT images for every patient by using the automatic window-level and width-setting system. As a result of this experiment, there was a considerable difference in window setting by an expert technologist and that by a beginner technologist. With our system, we were able always to obtain an optimal window setting, such as that set by an expert technologist, regardless of the CT experience of the radiological technologist. We think that this system will be effective in observing animated examinations even if film is no longer used in the future.

The use of CT as a general examination has spread widely and is even used in small institutions. However, it is difficult to determine the current situation of each institution. Therefore, we employed a questionnaire to investigate the current situation of a variety of institutions. From the results of the questionnaire, we determined that the window setting was difficult for beginner technologists. In addition, in many institutions, radiological technologists did not always use the same display FOV for the same patient. From this questionnaire, we were able to determine the present conditions in each institution. We consider these results very useful.

The purpose of this study is to investigate the feasibility of cardiac evaluation with a dynamic flat-panel detector (FPD), based on changes in pixel values during cardiac pumping. To investigate the feasibility of cardiac evaluation with a dynamic flat-panel detector (FPD), based on changes in pixel values during cardiac pumping. Sequential radiographs of a cardiac motion phantom and water-equivalent material step were obtained with an FPD system. Various combinations of cardiac output and heart rate were evaluated with and without contrast medium. The ventricular area and summation of pixel values in the ventricles were measured. The ejection fraction (EF) was calculated based on the rate of changes and then compared to EF obtained from computed tomography images. In addition, slight changes in pixel values were visualized by use of inter-frame subtraction and color-mapping. The result of a clinical case was examined according to cardiac physiology. There were strong correlations between EF and our results. There was no significant difference between the findings with and without contrast medium. When the heart rate was greater than 60 bpm, EF obtained with our method were underestimated. It is necessary for a patient to be examined at an imaging rate between 7.5 and 10 fps at least. In addition, a +/-1.2% change in pixel value was equivalent to a +/-10 mm change in the thickness of water. Color-mapping images were supported by cardiac physiology. Evaluating changes in pixel values on dynamic chest radiography with FPD has the potential to demonstrate cardiac function without contrast medium. Inter-frame subtraction and color-mapping are very useful for interpreting changes in pixel value as velocities of blood flow.

It is poorly understood how oxidized LDL (oxLDL) promotes monocyte dynamics in transendothelial migration (TEM) in atherogenesis. We developed an in vitro 3D-live-single cell TEM assay system with subendothelial oxLDL embedded in ultra-thin collagen gels, mimicking subendothelial oxLDL accumulation in vivo. With dividing monocyte dynamics into three stages (1: adhesion on endothelium, 2: invasion and 3: complete transmigration below endothelium), we analyzed the stage transition dynamics of individual living human monocytes. OxLDL did not enhance initial monocyte adhesion to endothelium (stage 1), but it specifically primed adherent monocytes to start invasion (stage 1-->2). Once invasion started, it had no effect thereafter on monocyte stage transition (stage 2-->3). OxLDL upregulated PECAM-1 and downregulated VE-cadherin on endothelial junctions without monocyte addition, both of which could promote monocyte entry by enhanced homophilic binding to monocyte PECAM-1, and by disrupted junctional barrier, respectively. Meanwhile, monocyte speed at neither locomotion on endothelium (stage 1) nor subendothelial migration (stage 3) was altered by oxLDL. These data indicate that before monocyte adhesion, endothelial junctions changed their conformation to more monocyte-acceptable state in response to oxLDL, resulting the stage-specific promotion of monocyte TEM (stage 1-->2; initiation of invasion) with no enhancement of its initial adhesion or migration speed.

Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or L-arginine and was partially reduced by the addition of diphenylene iodonium or N(G)-nitro-L-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2',7'-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker) with L-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (BH4), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of BH4 availability. These mechanisms are potential key targets for therapeutic interventions.

Dyschromatosis symmetrica hereditaria (DSH) (also called "reticulate acropigmentation of Dohi") is a pigmentary genodermatosis of autosomal dominant inheritance. We have clarified for the first time four pathological mutations of the double-stranded RNA-specific adenosine deaminase gene (ADAR1 or DSRAD) in four DSH pedigrees. In this paper, we report 16 novel mutations containing six missense substitutions (p.V906F, p.K1003R, p.G1007R, p.C1036S, p.S1064F, p.R1078C), two splice site mutations (IVS2+2T>G, IVS8+2T>A), six frameshift mutations (p.H216fs, p.K433fs, p.G507fs, p.P727fs, p.V955fs, p.K1201fs), and two nonsense mutations (p.R426X, p.Q600X) found in Japanese patients with DSH. We did not establish any clear correlation between the clinical phenotypes and the genotypes of ADAR1 gene mutations in our examination of 16 cases plus four pedigrees. None of the different mutations identified in our studies of 20 cases suggested any founder effect. Furthermore, we did not identify any mutations in the ADAR1 gene of three patients with dyschromatosis universalis hereditaria or three patients with acropigmentatio reticularis, indicating that the two diseases are completely different from DSH, although they have sometimes been suggested to be phenotypical variations of DSH.

We demonstrated recently that chronic administration of aldosterone to rats induces glomerular mesangial injury and activates mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK1/2). We also observed that the aldosterone-induced mesangial injury and ERK1/2 activation were prevented by treatment with a selective mineralocorticoid receptor (MR) antagonist, eplerenone, suggesting that the glomerular mesangium is a potential target for injuries induced by aldosterone via activation of MR. In the present study, we investigated whether MR is expressed in cultured rat mesangial cells (RMCs) and involved in aldosterone-induced RMC injury. MR expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. Cell proliferation and micromechanical properties were determined by [3H]-thymidine uptake measurements and a nanoindentation technique using an atomic force microscope cantilever, respectively. ERK1/2 activity was measured by Western blotting analysis with an anti-phospho-ERK1/2 antibody. Protein expression and immunostaining revealed that MR was abundant in the cytoplasm of RMCs. Aldosterone (1 to 100 nmol/L) dose-dependently activated ERK1/2 in RMCs with a peak at 10 minutes. Pretreatment with eplerenone (10 micromol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation and decreased the elastic modulus, indicating cellular proliferative and deforming effects of aldosterone, respectively. These aldosterone-induced changes in cellular characteristics were prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor, PD988059 (100 micromol/L). The results indicate that aldosterone directly induces RMC proliferation and deformability through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.

One of the newest CT application technologies is cardiac synchronized image reconstruction. In this technology, evaluation of time-resolution is very important. We developed a method of measuring time-resolution in cardiac synchronized reconstruction, and evaluated various scanning protocols. In our experiment, ECG-gated scanning was done by multi-slice CT (Aquilion16 Super Heart Edition, Toshiba Medical Systems Co., Ltd., Japan). The nominal slice thickness was 0.5 mm, and rotation time was 0.5 sec. Input heart rate was set at 40, 45, 50, 55, 60, 70, 75, 80, and 90 bpm, and helical pitch at 3.2, 4.0, and 4.8 (beam-pitch: 0.200, 0.250 and 0.300). We measured FWTM of the obtained sensitivity distribution and compared at each scanning protocol. Time resolution improved as helical pitch decreased and heart rate increased. However, phase-time resolution deteriorated as heart rate increased. The results of our experiment indicated that a segment center was determined by X-ray tube rotation time and heart rate, and the number of segments was determined by heart rate, helical pitch, and reconstruction position. Time resolution changed with X-ray tube rotation time, heart rate, helical pitch, and reconstruction position. In this report, we provide a reference for an optimal scanning protocol in cardiac synchronized image reconstruction.

Prodromal signs such as cardiac rhythm disturbance and changes in gastric motility are generally induced before and during nausea in humans. These autonomic reactions were compared in subjects who were or were not experiencing nausea. Nausea was induced by having the subjects view a movie of oscillating pictures. Seventeen healthy volunteers were asked to relax their muscles and watch the movie. Electrogastrogram (EGG), electrocardiogram (ECG), palmar and metopic perspiration, digital blood flow and thoracic movement related to respiration were simultaneously measured while the subjects viewed the movie. A total of 11 of 17 subjects complained of nausea after watching the movie. The characteristic changes in their autonomic responses during exposure to the movie were as follows. The power of the EGG, heart rate and metopic perspiration significantly increased compared to those before watching the movie. The respiratory cycle gradually increased during and even after watching the movie. In contrast, no significant changes in the power of the EGG, heart rate and metopic perspiration were observed in the remaining six subjects who did not experience nausea. The role of the autonomic nervous system in nausea is discussed. These results suggest that these symptoms regarding the sympathetic nervous system could actually be defensive reactions against the sensation of nausea.

Objective: To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM)., Methods and Results: We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30-50 microm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 +/- 5.4 min (mean +/- S.D., n=61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 +/- 3.2 min (mean +/- S.D., n=53 monocytes)., Conclusions: Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.

We examined the sarcomere length-dependence of the spacing of the hexagonal lattice of the myofilaments and the mass transfer of myosin cross-bridges during contraction of right ventricular papillary muscle of the rat. The lattice spacing and mass transfer were measured by using X-ray diffraction, and the sarcomere length was monitored by laser diffraction at the same time. Although the lattice spacing and the sarcomere length were inversely related, their relationship was not exactly isovolumic. The cell volume decreased by about 15% when the sarcomere length was shortened from 2.3 micro m to 1.8 micro m. Twitch tension increased with sarcomere length (the Frank-Starling law). At the peak tension, the ratio of the intensity of the (1,0) equatorial reflection to that of the (1,1) reflection was smaller when the tension was greater, showing that the larger tension at a longer sarcomere length accompanies a larger amount of mass transfer of cross-bridges from the thick to the thin filament. The result suggests that the Frank-Starling law is due to an increase in the number of myosin heads attached to actin, not in the average force produced by each head.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.

We studied x-ray diffraction from the left ventricular wall of an excised, perfused whole heart of a rat using x rays from the third-generation synchrotron radiation facility, SPring-8. With the beam at right angles to the long axis of the left ventricle, well-oriented, strong equatorial reflections were observed from the epicardium surface. The reflections became vertically split arcs when the beam passed through myocardium deeper in the wall, and rings were observed when the beam passed into the inner myocardium of the wall. These diffraction patterns were explained by employing a layered-spiral model of the arrangement of muscle fibers in the heart. In a quiescent heart with an expanded left ventricle, the muscle fibers at the epicardium surface were found to have a (1,0) lattice spacing smaller than in the rest of the wall. The intensity ratio of the (1,0) and (1,1) equatorial reflections decreased on contraction with a similar time course in all parts of the wall. The results show that it is possible to assign the origin of reflections in a diffraction diagram from a whole heart. This study offers a basis for interpretation of x-ray diffraction from a beating heart under physiologically and pathologically different conditions.

Intra-aortic measurement of nitric oxide (NO) would provide valuable insights into NO bioavailability in systemic circulation and vascular endothelial function. In the present study, we thus developed a catheter-type NO sensor to measure intra-aortic NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter to measure intra-aortic plasma NO concentration in vivo in anesthetized dogs. Infusion of acetylcholine (10 microg/kg) increased base-to-peak plasma NO level in the aorta by 2.4+/-0.4 nM (n=7). After 20-min infusion of N(G)-methyl-L-arginine (NO synthase inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8+/-0.4 nM, P<0.003, n=7). In conclusion, the newly developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo and demonstrated applicability to direct evaluation of intravascular NO bioavailability.

The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-to-substrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1beta-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage.

Direct measurement of nitric oxide (NO) is of great importance and value for both in vitro and in vivo studies on dynamic NO bioactivity. Here, we evaluated the basic performance of a newly developed NO sensor (Innovative Instruments, Inc.). Unlike other NO sensors, the new NO sensor has a highly durable, gas-permeable coating and is affected much less by electrical interference due to its integrated structure where working and reference electrodes are combined in a single element. Calibration with NO gas showed high sensitivity of about 580 pA per nmol-NO l(-1) (the detection limit 0.08 nmol-NO l(-1), S/N = 3). This sensor also showed high selectivity (25,000 times and more) to NO, compared with NO-related reagents such as L-arginine, N(G)-monomethyl-L-arginine, acetylcholine, nitroglycerin (NTG) and tetrahydrobiopterin as well as dissolved oxygen. As an in vivo application, the sensor was located in the anaesthetized rat abdominal aorta to measure NTG-derived plasma NO. lntra-aortic infusion of 0.5 mg NTG caused a measurable increase in plasma NO level (2.0 +/- 2.2 nmol l(-1), mean +/- SD, n = 3). In conclusion, the new NO sensor demonstrated a satisfying performance for both in vitro and in vivo applications.

We examined the effects of brovincamine fumarate, a Ca(2+)-channel blocker, on choroidal blood flow. We measured the choroidal blood volume continuously for 1 hour using laser Doppler flowmetry, as well as systemic blood pressure, heart rate, and intraocular pressure in six urethane-anesthetized rabbits after intravenous administration of 0.1 mg/kg or 0.5 mg/kg brovincamine. As a control, ten rabbits receiving no medication were used. All the data were recorded and analyzed using MacLab on a computer. In both the 0.1 mg/kg and 0.5 mg/kg brovincamine-injected groups, the choroidal blood volume decreased significantly after administration, but showed no significant difference from controls. Vascular resistance in the choroid showed a significant increase over the value before administration and over the control group. The heart rate decreased significantly compared to the value before injection and to the control group. The mean blood pressure in both dose groups and the intraocular pressure in the 0.5 mg/kg injected group were significantly higher than the controls. These results indicate that intravenous administration of 0.1 mg/kg or 0.5 mg/kg brovincamine does not cause an increase in the choroidal blood volume in urethane-anesthetized rabbits.

1. To evaluate the effects of cardiac contraction on intramyocardial (midwall) microvessels, we measured the phasic diameter change of left ventricular intramural arterioles and venules using a novel needle-probe videomicroscope with a CCD camera and compared it with the diameter change in subepicardial and subendocardial vessels. 2. The phasic diameter of the intramural arterioles decreased from 130 +/- 79 ìm in end-diastole to 118 +/- 72 micron (mean +/- S.D.) in end-systole by cardiac contraction (10 +/- 6 %, P < 0.001, n = 21). 3. The phasic diameter in the intramural venules was almost unchanged from end-diastole to end-systole (85 +/- 44 vs. 86 +/- 42 micron, respectively, 2 +/- 6 %, n. s., n = 14). 4. Compared with intramural vessels, the diameters of subendocardial arterioles and venules decreased by a similar extent (arterioles: 10 +/- 8 %, P < 0. 001; venules: 12 +/- 10 %, P < 0.001) from end-diastole to end-systole, respectively, whereas the diameter of the subepicardial arterioles changed little during the cardiac cycle, and subepicardial venule diameter increased by 9 +/- 8 % (P < 0.01) from end-diastole to end-systole. These findings are consistent with our previous report. 5. We suggest that the almost uniform distribution of the cardiac contractility effect and arteriolar transmural pressure between the subendocardium and the midmyocardium, which together constitute the systolic vascular compressive force, accounts for the similarity in the arteriolar diameter changes in both myocardial layers. The smaller intravascular pressure drop from deep to superficial myocardium relative to the larger intramyocardial pressure drop explains the difference in the phasic venular diameter changes across the myocardium.

There is a paradoxical alpha-adrenoceptor-mediated coronary vasoconstriction whenever there is adrenergic activation of the heart, as during cardiovascular reflexes or exercise. A previous study demonstrated that this paradoxical vasoconstriction helps maintain blood flow to the vulnerable inner layer of the left ventricular wall during exercise, but the mechanism for this effect was not elucidated. The purpose of the present investigation was to test the hypothesis that alpha-adrenoceptor-mediated vasoconstriction lessens the to-and-fro oscillation of blood flow that occurs in the coronary arterial tree during systole and diastole. Septal coronary artery blood velocity was measured in anesthetized open-chest dogs with a 20-MHz pulsed Doppler velocimeter. Systolic retrograde velocity and diastolic forward velocity were compared during norepinephrine infusion before and after alpha-adrenoceptor blockade with phenoxybenzamine. Systolic aortic pressure was held constant by aortic banding; heart rate was controlled by pacing at 80, 140, and 200 beats/min; and maximum left ventricular dP/dt was unchanged by alpha-blockade. At each pacing rate, systolic retrograde velocity was significantly greater after alpha-blockade, indicating that alpha-vasoconstriction reduced systolic retrograde flow by changing coronary vascular impedance. Transmural blood flow was measured with microspheres in a second group of dogs during the same experimental conditions, and flow to the inner layer of the left ventricle was diminished by alpha-adrenoceptor blockade at a heart rate of 250 beats/min, demonstrating a beneficial effect of alpha-vasoconstriction. In conclusion, adrenergic alpha-adrenoceptor-mediated coronary vasoconstriction reduces systolic retrograde coronary flow during norepinephrine infusion. This lessens to-and-fro flow oscillation in the coronary circulation and probably is the mechanism whereby alpha-vasoconstriction helps maintain blood flow to the inner layer of the left ventricle during exercise.

Objectives: We sought to evaluate the effect of intraaortic balloon pumping on the phasic blood velocity waveform into myocardium with severe coronary artery stenosis., Background: In the presence of severe coronary artery stenosis, it is not clear whether intraaortic balloon pumping augments intramyocardial inflow during diastole or changes systolic retrograde blood flow from the myocardium to the extramural coronary arteries., Methods: Using anesthetized open chest dogs (n=7), we introduced severe stenosis in the left main coronary artery to reduce the poststenotic pressure to approximately 60 mm Hg (>90% diameter stenosis). Septal arterial blood flow velocities were measured with a 20-MHz, 80-channel ultrasound pulsed Doppler velocimeter. Left anterior descending arterial flow, aortic pressure and poststenotic distal coronary pressure were measured simultaneously. The diastolic anterograde flow integral and systolic retrograde flow integral were compared in the presence and absence of intraaortic balloon pumping., Results: Although intraaortic balloon pumping augmented diastolic aortic pressure, this pressure increase was not effectively transmitted through stenosis. Septal arterial diastolic flow velocity was not augmented, and left anterior descending arterial flow was unchanged during intraaortic balloon pumping., Conclusions: In the presence of severe coronary artery stenosis, intraaortic balloon pumping failed to increase diastolic inflow in the myocardium and did not enhance systolic retrograde flow from the myocardium to the extramural coronary artery. Thus, the major effect of intraaortic balloon pumping on the ischemic heart with severe coronary artery stenosis may be achieved by reducing oxygen demand by systolic unloading.

Our aim was to evaluate the effects of intraaortic balloon pumping (IABP) on the blood velocity waveform in the absence or presence of coronary artery stenosis. Using anesthetized open-chest dogs, the septal arterial blood flow velocities were measured with a 20 MHz 80-channel ultrasound pulsed Doppler velocimeter in the absence (n = 5) or presence (n = 3) of left main coronary artery stenosis. The blood velocity waveform was analyzed by calculating the systolic retrograde velocity integral (SR) and the diastolic antegrade velocity integral (DA). A slosh ratio was defined as SR/DA. The left anterior descending arterial flow (CBF), aortic pressure (AoP), and poststenotic distal coronary pressure (DiP) were also measured simultaneously. We compared the effect of IABP on the velocity waveforms in the absence and in the presence of coronary artery stenosis. In the absence of stenosis, IABP increased DiP during diastole and augmented DA while it also increased SR. IABP augmented the net CBF because of the greater increase in DA than SR. In the presence of stenosis, however, IABP did not increase DiP and resulted in no significant effect on the net CBF.

Blood velocity profiles were measured in the renal branch (diameter 5.9 +/- 1.3 mm) of the aortorenal bifurcation using a 20-MHz 80-channel pulsed Doppler velocimeter during retroperitoneal surgery in 10 patients. The peak Reynolds number was 1145 +/- 140 and the frequency parameter (Wormersley parameter) was 3.0 +/- 0.8. Immediately distal to the ostium of the renal artery, reverse flow, indicating flow separation, was observed near the cranial wall mainly during the first part of the cardiac cycle. There were flows from the cranial to the caudal side of the artery at this location, indicating the presence of strong secondary flows. Two diameters downstream of the ostium, the velocity profiles were skewed to the caudal side in all patients. Four diameters downstream, the flow profile was symmetrical (3 patients) or only slightly skewed (7 patients) and virtually parabolic throughout the cardiac cycle. These observations mean that the flow on the cranial side of the renal branch of the human aortorenal bifurcation is characterized by (1) a bidirectional oscillation of the flow, (2) separation of the flow during systole, and (3) low time-averaged shear rate. These blood velocity patterns may be related to the localization and development of atheromatous plaque that occurs preferentially in this region of the renal artery. Conversely, the unidirectional, axisymmetrical flow found in more distal parts of the renal artery are associated with a very low incidence of lesions.

To study the vasodilatory capacity of subendocardial (ENDO) arterioles, we evaluated the reactive hyperemic responses of ENDO as well as subepicardial (EPI) arterioles in 40 dogs by our needle-probe intravital microscope. We also examined the individual and combined effects of an ATP-sensitive K+ channel blocker (glibenclamide, 200 micrograms/kg), an inhibitor of nitric oxide synthase (NG-monomethyl-L-arginine [L-NMMA], 2 mumol/min, 20 minutes), and an adenosine-receptor antagonist (8-phenyltheophylline [8PT], 0.75 mumol/min, 15 minutes). The percent increase in end-diastolic diameter of ENDO arterioles was larger (P < .01) than that of EPI arterioles during reactive hyperemia, especially for the arterioles larger than 120 microns (P < .01). The diastolic-to-systolic vascular pulsation amplitude at the peak flow was greater in ENDO than EPI arterioles (25% versus 6%, P < .05). Compared with control conditions, the presence of both glibenclamide and L-NMMA suppressed the vasodilation responses of ENDO arterioles (P < .01 for both) and EPI arterioles (P < .05 for both). The effect of L-NMMA was greater in ENDO arterioles (P < .01), but that of glibenclamide was not different between ENDO and EPI arterioles. 8PT influenced the hyperemic response, although statistical significance was found only in the flow response. The effect of combined infusion of L-NMMA and glibenclamide with or without 8PT was greater than that of individual infusions in both ENDO and EPI arterioles. Conclusions are as follows: (1) The vasodilatory response of ENDO arterioles was even larger than that of EPI arterioles. Thus, the smaller flow reserve of ENDO arterioles may be caused by other factors, including the greater effects of myocardial compression and nitric oxide on the ENDO arterioles. (2) The vascular responses of ENDO and EPI arterioles were modulated by both endothelium-independent and -dependent vasodilative factors, and the effect of each factor including adenosine was associated with the effects of others.

The purpose of this study was the visualization of coronary arteries by the diastolic reconstruction method with helical scanning (HES) CT. We chose diastolic phase images from the image data acquired with the HES technique using a continuous nutate-rotate fast CT scanner, because few motion artifacts are induced by cardiac pulsation during the slow filling diastolic phase. We assessed coronary diseases using this method based on HES-CT, and evaluated its clinical effectiveness. We also studied the clinical value of multiplanar reconstruction and three-dimensional surface reconstruction. The subjects consisted of 31 patients with coronary disease, aged 41-77 years. When a diastolic reconstruction HES-CT was employed, the average rate of visualization of the coronary arteries was 72%. An examination can be completed during a 30-second single breath-hold, permitting the acquisition of coronary artery images with excellent continuity. Calcified coronary arteries can be identified.

Helical scanning CT (HES-CT) was applied to multiplanar reconstruction (MPR) and 3-D imaging for the diagnosis of 65 patients with orbital lesions. We employed the the Toshiba TCT-900S, a fourth-generation high-speed CT scanner equipped with a slip-ring system. In HES-CT, scanning was performed with continuous rotation of the X-ray tube for 30 s, while the couch top was simultaneously moved at a constant speed of 1 to 2 mm/s. Images were reconstructed from continuous raw data to obtain a slice pitch of 1 mm in the longitudinal direction. HES-CT was found to have some advantages over conventional thin slice CT: 1) a wide area could be scanned in a short time (30 s) without motion artifact, 2) small lesions could be easily detected because slice images could be generated with a fine pitch at arbitrary positions from continuous raw data, 3) MPR images had good continuity in the longitudinal direction, and 4) high contrast of structures was obtained in the orbital region. HES-CT was considered to be useful for the detection of orbital lesions.

Direct and continuous observation of subendocardial (deep myocardial) microcirculation provides essential information on coronary circulation, since cardiac contraction affects subendocardial vessels most vigorously. To achieve this aim, we developed a portable needle-probe video-microscope with a charge-coupled-device (CCD) camera to visualize the subendocardial microcirculation. Images of the subendocardial microcirculation of a porcine beating heart were successfully observed in all cases. The vascular compression by cardiac contraction decreased the diameter of subendocardial arterioles and venules by about 20%.

This study was carried out to investigate the characteristics of coronary arterial flow in left ventricular hypertrophy secondary to systemic hypertension. The blood velocities in the left anterior descending coronary artery (LAD) were measured by a No. 3F 20 MHz Doppler catheter in 23 hypertensive patients with left ventricular hypertrophy (systolic/diastolic pressure: 181 +/- 15/100 +/- 4 mmHg) and 13 patients with atypical chest pain, but without left ventricular hypertrophy and any abnormal hemodynamic findings. All patients had normal coronary arteriograms. The LAD blood velocity waveforms in pressure overloaded left ventricular hypertrophy were characterized by both a decreased mid-to-late diastolic deceleration rate (delta V/delta T) and a normalized value of delta V/delta T by peak diastolic velocity [delta V/(delta T.Vpeak)], as well as delayed early diastolic inflow (time for diastolic rise; TDR). The values of the delta V/(delta T.Vpeak) in the patients with hypertensive left ventricular hypertrophy and in the normotensive controls were 1.26 +/- 0.61 and 3.03 +/- 1.18/s, respectively (P < 0.001). The TDR was 145 +/- 56 and 66 +/- 15 ms (P < 0.001). In patients with hypertensive left ventricular hypertrophy, the delta V/(delta T.Vpeak) correlated well with the degree of hypertrophy (r = 0.75, P < 0.01) and with the TDR (r = 0.82, P < 0.01). The coronary flow reserve, calculated from the ratio of the diastolic mean velocity after intracoronary injection of papaverine to the resting flow velocity increased with the delta V/(delta T.Vpeak) (r = 0.68, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated spatial resolution in the longitudinal axis with helical scanning CT using a fourth-generation fast CT scanner. We made a phantom by stringing acrylic balls (65 mm phi x 8 and 9 mm phi x 6). The acquired images were processed by MPR and assessed visually to evaluate axis resolution. With the conventional scanning method, the partial volume effect varied with the starting position, but helical scanning was able to reconstruct high-resolution images using continuous raw data. During helical scanning, axis resolution varied depending on the slice width and sliding speed of the couch top. Even if the sliding speed was kept constant at 4 mm/sec, axis resolution was superior with a slice width of 2 mm than with one of 5 mm.

The murine temperature-sensitive cell-cycle mutant, ts85, shows an abnormal induction of heat-shock proteins which is different from the wild type FM3A cells. This paper explores the effect of culture temperature on the expression of heat shock proteins in ts85 cells. When ts85 cells were maintained at 33 or 37 degrees C, these cells synthesized heat-shock protein (hsp) 70 following continuous heating at 39 degrees C or subsequent incubation after heating at 42 degrees C for 15 min. In contrast, these conditions are not conducive for hsp70 synthesis by FM3A cells. Moreover, ts85 cells which were maintained at 37 degrees C synthesized hsp70 following continuous heating at 42 degrees C or subsequent incubation after heating at 45 degrees C for 15 min. The synthesis of hsp70 in these cells corresponded to an increased level of hsp70 mRNA. Furthermore, the constitutive hsp105 level of cells maintained at 33 degrees C was only half of that of cells which were maintained at 37 degrees C, and cells maintained at 33 degrees C were more sensitive to subsequent heat treatment than those maintained at 37 degrees C. These results indicate that culture temperature not only affects the induction of hsp70 mRNA, but also cellular levels of hsp105 and the resulting thermal sensitivity of ts85 cells. These findings suggest that the other phenotypic characteristic of the mutant ts85 cells is also affected by culture temperature.

Using a 20-MHz 80-channel pulsed Doppler velocimeter and 30-MHz high-resolution echo ultrasound, we investigated the in vivo hemodynamics at the origin of the renal artery by measuring the velocity profiles and bifurcation geometry of a surgically exposed left renal artery in 10 anesthetized dogs. The angle between the aorta and the renal artery ranged from 60 degrees to 90 degrees (mean, 84 degrees) although the bifurcation did not lie in a single anterodorsal plane and the diameter of the renal artery ranged from 1.5 to 3.5 mm (mean, 2.4 mm). Despite different geometries, the velocity profiles in the different aortorenal bifurcations were similar. Although regions of reverse velocity were observed, the net flow in the renal artery was in the forward direction throughout the cardiac cycle. The peak Reynolds' number was 486 +/- 63. The velocity profiles in the proximal renal artery in the plane parallel to the bifurcation showed velocity vectors directed toward the caudal wall throughout the cardiac cycle. Reverse flow, indicating flow separation, was observed near the cranial wall even during systole. When the probe was placed on the cranial wall perpendicular to the wall, a velocity component from the cranial side to the caudal side was observed. At a distance of four diameters from the renal ostia, velocity profiles were almost parabolic. These results indicate that the velocity pattern near the cranial wall at the renal ostia, at which atherosclerotic lesions are prone to develop, are characterized by 1) a low time-averaged shear rate, 2) separation of the flow, and 3) a time-varying oscillation of the flow.

We have shown that heat shock does not induce the synthesis of hsp70 in FM3A cells maintained at a low culture temperature of 33 degrees C although it does so in cells maintained at 37 degrees C [T. Hatayama et al. (1991) Biochem. Int. 24, 467-474]. In this paper, we show that FM3A cells maintained at 37 degrees C produced hsp70 mRNA during continuous heating at 42 degrees C or during postincubation at either 37 or 33 degrees C after being heated at 45 degrees C for 15 min, whereas cells maintained at 33 degrees C did not produce hsp70 mRNA during continuous heating at 37, 39, 42, or 45 degrees C, or during postincubation after being heated at any temperature. Thus the lack of hsp70 synthesis in cells maintained at 33 degrees C seemed to be due to the absence of hsp70 mRNA induction. Also, hsp70 was accumulated in cells maintained at 37 degrees C during continuous heating at 42 degrees C and during postincubation at 37 degrees C after heat shock at 45 degrees C, but not during postincubation at 33 degrees C. The cellular level of the constitutive hsp73 as well as the mRNA level were both similar in cells maintained at 33 and 37 degrees C. On the other hand, the cellular level of the constitutive hsp105 in cells maintained at 33 degrees C was only half of that in cells maintained at 37 degrees C. These hsp105 levels increased significantly in both types of cells after continuous heating at 39 degrees C. These findings indicate that the culture temperature affects not only the induction of hsp70 mRNA but also the accumulation of hsp70 and hsp105 in the cells.

Hemangiomatous tumors were induced in Balb/c nude mice by inoculating F-2 cells (5 x 10(6)) from a novel tumorigenic murine endothelial cell line which had been established and maintained in our laboratories. These tumors were morphologically investigated in the course of development. Subcutaneous hemorrhage was observed at the inoculation site within 12 hours after the injection of F-2 cells, followed by development of skin tumors of various sizes at the same sites. They were dome-shaped, glossy surfaced, black, soft tumors. Mice finally died of massive blood loss due to internal and/or external hemorrhage (on day 10-77). Light microscopically, F-2 cells formed aggregates immediately after inoculation and then branched into a network of channels and cysts containing erythrocytes. Thereafter, spongiform structures composed of various sizes of cysts appeared with subsequent formation of a single large blood-filled cyst lined by one or two layers of thin cells. Under an electron microscope, F-2 cells, possessing large amounts of cytoplasm, formed narrow spaces which were occasionally incomplete with ambiguous basal lamina at the early stages. However, later, in large cysts, they became attenuated, tightly connected, and produced complete lumen surrounded by a basal lamina. Immunohistological demonstration of H-2 K, D antigen showed that tumors induced by F-2 cells mainly consisted of the inoculated F-2 cells. These results indicate that F-2 presents a good experimental system for investigation of vascular endothelial cell tumorigenesis and differentiation.

Effects of low culture temperature on the induction of heat shock proteins in FM3A cells by a heat shock and on the thermal sensitivity of the cells were examined. FM3A cells maintained at 33 degrees C could not induce hsp70 during continuous heating or after a short heat shock at either 39, 42, or 45 degrees C, although FM3A cells maintained at a normal culture temperature of 37 degrees C can induce the synthesis of hsp70. Furthermore, the cells maintained at 33 degrees C were more sensitive to the subsequent heat shock than the cells maintained at 37 degrees C. Thus, the culture temperature of the mammalian cells may be an important factor for the induction of hsp70, and hsp70 may play an important role to protect or repair the thermal damage of cells.

To clarify the characteristics and possible causes of phasic blood velocity patterns in pressure overload hypertrophy, we measured blood flow velocities in the left anterior descending coronary arteries in 16 patients with left ventricular hypertrophy secondary to systemic hypertension. These measurements were made with a 20MHz Doppler catheter. All patients had normal coronary arteriograms. The blood flow velocity patterns were characterized by the decrease in the rise of early diastolic inflow velocity. Prolongation in the time from the onset of diastole to peak velocity (TDPV) correlated with the degree of left ventricular hypertrophy. TDPV was prolonged in proportion to the increase in the peak left ventricular systolic pressure and left ventricular ejection fraction. The flow reserve calculated from the ratio of the diastolic mean velocity after the intracoronary injection of papaverine to the resting flow showed a trend toward the decrease proportional to the prolongation in TDPV due to an increase of wall thickness. In conclusion, preceding systolic vascular compression and systolic vascular strain can be factors causing early diastolic inflow disturbance in left ventricular hypertrophy secondary to hypertension. The decrease of coronary reserve may partially be attributable to the decrease in the rise of early diastolic inflow velocity.

A new murine cell line, designated F-2, was established from an ultraviolet light-induced tumor which developed on the back skin of a BALB/c x C57BL/6 F1-nu/nu nude mouse. More than 1 x 10(5) F-2 cells injected into nude mouse skin produced rapidly developing hemangiomatous lesions. F-2 had a 14-h doubling time under 10% fetal calf serum-containing Dulbecco's modified Eagle's medium culture conditions without any cell growth factor supplementation, and it showed "cobblestone" appearance at confluency. F-2 was able to rapidly differentiate on Matrigel with resultant fine network structure and tubule formation. Ultrastructural observations of the tubule demonstrated that the F-2 cells were connected to each other by intermediate junctions and arranged to form spaces, but that no Weibel-Palade bodies were found in the cytoplasm. F-2 also showed the active uptake of fluorescent-labeled acetylated low-density lipoprotein at 37 degrees C but not at 4 degrees C, and it showed prominent binding to the lectin, Griffonia simplicifolia I agglutinin. The addition of fibroblast growth factor did not facilitate the growth of F-2 cells. These findings strongly suggest that F-2 is a transformed cell line with tumorigenicity and vascular endothelial cell properties, and it may be useful in the study of vascular tumor biology.

Sweet's syndrome arose in a 54-year-old Japanese man who had suffered from general malaise, episodic fever, and lymphadenopathy for ten months. Skin symptoms subsided rapidly, but he died three months later with a postmortem diagnosis of immunoblastic lymphadenopathy.

The accuracy of 20-MHz Doppler catheter coronary artery velocimetry (Millar Instruments) was evaluated by model experiments using a turntable and a flow tube. In the turntable experiments, the flow away from the catheter tip (away flow) was significantly lower than that toward the catheter (toward flow), but the measured velocities for both away and toward flows were always lower than the known velocities. Nevertheless, the blood velocities measured at a sampling point 4 mm from the catheter tip showed excellent correlation with the known velocities irrespective of flow direction. The shape of the FFT spectra of the Doppler shift frequency from blood was broadened, and the frequency corresponding to the known velocity was always close to the maximum Doppler shift frequency. We concluded that zero-cross detection of blood velocity by the 20-MHz pulsed Doppler catheter provides a reliable measure for evaluating relative velocity changes, although it underestimates the blood velocity.